Method for the production of desiccation tolerant plant embryoids and methods for germination of desiccation tolerant embryoids

ABSTRACT

A method for the induction of desiccation tolerance in plant embryoids is disclosed. The method entails using an excess of absissic acid and a coating of apolar and polar hygroscopic material.

The invention relates to methods for the induction of desiccationtolerance in plant embryoids. It also relates to methods for thegermination of embryoids which have been dessicated. Novel desiccatedembryoids are also provided.

Regeneration of storable embryoids has been described for a large numberof species. Redenbaugh et al. (1986) were the first to create artificialseeds by encapsulation of embryoids in alginate gel beads. However, thestill hydrated artificial seeds were difficult to store, because theylacked quiescence, and the conversion rate (outgrowth into plantlets)was very low. The recent achievement of induction of tolerance tocomplete desiccation (Senaratna et al., 1989a) may give newopportunities to the artificial seed technology. Desiccated embryoidsare better suited for use in this technology, because they are in aquiescent state comparable to that of dry seeds and they therefore havebetter storage properties. Gray (1990) even stated that dried grapeembryoids germinated better than fresh ones.

Desiccation tolerance is defined by us as the ability of embryoids toregrow after storage at 25° C. for two weeks under low moistureconditions (e.g. 10%, comparable to dry seeds).

Production of desiccation tolerant embryoids has been published for thefollowing species: alfalfa (Anandarajah and McKersie, 1990 and 1991;Senaratna et al., 1989a,b; McKersie et al., 1989), geranium (Marsolaiset al., 1991), soybean (Parrott et al., 1988), spruce (Roberts et al.,1990; Attree et al., 1991), grape (Gray, 1990) and carrot (Lecouteux etal., 1992; Iida et al., 1992). In most studies the plant hormoneabscisic acid (ABA) was used in amounts equivalent to the amounts usedfor inducing quiescence to induce desiccation tolerance. By adding theappropriate amounts of ABA to the culture medium at the right stage ofdevelopment, depending on species and genotype, embryoids could bedehydrated to moisture contents of less than 10% (on a dry weight basis)with retention of some viability. Through the addition of ABA, it waseven possible to induce desiccation tolerance in callus cultures ofcarrot (Nitzsche, 1980) and Craterostigma plantagineum (Bartels et al.,1990). Koornneef et al. (1989) and Meurs et al. (1992) haveunequivocally demonstrated the role of endogenous ABA during seeddevelopment by using recombinants of ABA deficient and ABA insensitivemutants of Arabidopsis thaliana.

Not only exogenously supplied ABA, but also several stress treatmentscan induce the capacity to survive dehydration (Anandarajah andMcKersie, 1990 and 1991). However, stress treatments such as heat orosmotic shock through high sucrose concentrations, may raise theendogenous ABA levels (Skriver and Mundy, 1990) and might thus inducedesiccation tolerance.

The capacity of the embryoids to survive dehydration is also dependenton the drying method. Only Senaratna et al. (1989) gave a well defineddescription of the drying method, including drying rates and finalmoisture content in alfalfa embryoids.

Hoekstra et al. (1989) showed that regrowth of initially viable, dryorganisms is impaired because of imbibitional damage. In all theprevious studies on induction of desiccation tolerance in embryoids theplant recovery rates were always less than 100%. Poor embryoid quality,caused by less then optimal protocols, or asynchronic embryoiddevelopment could be the reason for the low recoveries.

The problem therefore remains to provide desiccated storable plantembryoids which are essentially 100% capable of germination afterprolonged storage.

The invention provides such embryoids.

Also a method for germination of such embryoids in a manner thatessentially all the embryoids germinate is provided.

The invention provides this solution through a method for the inductionof essentially complete desiccation tolerance in plant embryoids whereinthe embryoids are treated with an amount of abscisic acid activity whichis significantly above the amount used to induce quiescence.

The physiological function ascribed to abscisic acid is the induction ofquiescence. It has now been found that treatment of plant embryoids,especially in the torpedo stage, with a significantly higher amount ofabscisic acid activity, will induce an essentially complete desiccationtolerance. To induce completely desiccation tolerant embryoids togerminate for 100%, this treatment has also proven very advantageous.

The abscisic acid activity can be provided by exogenously administeredabscisic acid, but the activity can also (partly) be provided in situ.Methods to induce the production of abscisic acid in situ include, butare not limited to, temperature treatments, meaning heat shock (exposureto temperatures of 30-50° C.) and cold shock (0-10° C.), osmotic stress(exposure within the range of -0.5 to -2.5 mPa) using different salts,carbohydrates or polymers such as polyethylene glycol.

Suitable quantities of abscisic acid activity expressed in amounts ofabscisic acid are from approximately 110% of the amount needed to inducequiescence to approximately 1000%, preferable from about 110-500%, mostprefereable from about 120-200%, of the amount needed to inducequiescence. For carrots the amount to induce quiescence is about 3.8 μM.

Instead of in situ induction of part of the abscisic acid activity orapplying exogenous abscisic acid, it is of course possible to useabscisic acid analogs such as those described in Walton ("Abscisic acid:F. A.Addicott ed., academic press, 1983, chapter 4). These analogs maybe active themselves, but it is also possible that they are metabolizedto abscisic acid.

The methods for induction of desiccation tolerance are most preferablyapplied to embryoids which are in the so called torpedo stage. Althoughnot impossible, the induction of desiccation tolerance at other stagesis much less efficient. For carrots the torpedo stage seems essential.In order to succeed in an essentially 100% germination of desiccationtolerant embryoids, it is preferred that the embryoids are prehydratedbefore imbibition. This prehydration prevents the damage which mayresult from a too rapid uptake of water. A suitable prehydration can beprovided by exposing the plant embryoids to moist air with a relativehumidity of 100% for at least 2 hours at 25° C. Preferably at thistemperature, the treatment should not be longer then about 8 hours,because that will result in loss of viability due to a variety ofreasons, for instance phase transition of the membranes. At lowertemperatures the prehydration treatment may be longer.

An alternative to the prehydration step is imbibing the embryoids at atemperature above the transition temperature (Tm) of the phospholipidmembranes of the embryoids (25-50° C.). Tm is the temperature at whichtransition takes place of membrane phospholipids from the gel phase tothe liquid crystalline phase and vice versa. Another possibility is toenvelop the embryoids in a coating that is capable to regulate waterflow, e.g. an apolar artificial coating such as paraffin. The coatingshould, besides being at least partially permeable for water, bepermeable for oxygen and, of course, non-toxic. We have developedsuitable coatings for embryoids produced according to the invention,which coatings enable the production of storable artificial seeds, whichare capable of germination for essentially 100%. The coating methods andmaterials as well as the coated embryoids are also part of theinvention. To ensure that the embryoids rehydrate slowly when imbibed,the coatings must have some, be it minor, water permeability. Suitablecoatings therefor comprise apolar, preferably wax-like materials, suchas paraffin or stearin and the like. In order to provide the waterpermeability a more polar material has to be mixed with the wax-likematerial. Preferably this is a hygroscopic (inorganic) material such asclays or pumice, although other materials such as cellulose(derivatives) may be used.

Phase transition of the membranes can also be prevented by the use oftrisaccharides such as trehalose, sucrose or umbelliferose, possibly aspart of the coating.

Also important for a desiccation protocol which results in the inventedembryoids, is the dehydration rate. When the dehydration rate is toohigh, this will result in embryoids uncapable of germination, because ofdamage to the membranes (segragation or other unwanted events mayoccur).

When the dehydration rate is too low the embryoids may turn brown anddie before they are competely dry.

Suitable drying rates depend on the species of the embryoids, butgenerally speaking will be between 0.01 g H₂ O/g dry weight per hour and1 g H₂ O/g dry weight per hour, preferably between 0.01 and 0.5 g H₂O/g, most preferably between 0.01 and 0.1 g H₂ O/g dry weight per hour.For carrots the optimum drying rate is about 0.03 g H₂ O/g dry weightper hour. A person skilled in the art will be able to arrive at suitabledrying rates for other species. Other species that will be usable in themethods and will lead to products according to the invention are knownto the person skilled in the art. They include, but are not limited to,cucumber, melon, celery, pelargonium, beans, peas, alfalfa, etc.

The invention will be illustrated in more detail in the followingexperimental part.

MATERIAL AND METHODS

Plant Material

Two Daucus carota L . genotypes were used with entirely differentgenetic backgrounds. One is a commercial variety cv "Trophy" and theother a breeding line "RS 1". Seeds and cell suspension cultures of cv"Trophy" were kindly provided by Dr S. de Vries of the Department ofMolecular Biology, Agricultural University Wageningen. Seeds of "RS 1"were obtained from Royal Sluis, Enkhuizen, The Netherlands.

Media Preparation and Culture Conditions

All culture media were based on the Gamborg's B5 basal composition(Gamborg, 1968). Before autoclaving, the pH was adjusted to 5.8. Themedia were sterilized for 20 minutes at 121° C. However, ABA wasdissolved in 0.2% NaHCO₃ and filter-sterilized (0.2 μm pore sizedisposable filter) before addition to the cooled medium. The cultureswere grown in a climate chamber with a 16 h/day photoperiod andcontinuous temperature of 25° C.

Suspension Culture

After surface sterilization with 2% NaOCl (20% commercial bleachsolution), the seeds were germinated on solid B5 medium (8 g/l agar).Sterile hypocotyl explants of ten day old seedlings were used to produceviable callus on solid B5 medium supplemented with 2.3 μM 2,4-D and 20g/l sucrose (later referred to as 2,4-D-B5). Cell suspension cultureswere started with 1 g callus per 50 ml 2,4-D-B5 medium in 250 mlErlenmeyer flasks on a rotary shaker at 100 rpm. The suspensions weremaintained by subculturing 2 ml PCV (packed cell volume) in 50 ml freshmedium, every 14 days. Seven days after refreshing, the cell suspensionswere used to regenerate embryoids.

Embryoid Production

Regeneration of embryoids occurred after transfer of the proembryogenicmasses (PEMS) to 2,4-D-free B5 medium with 20 g/l sucrose (OB5) at lowdensity (approximately 30,000 cells/ml) (De Vries et al., 1988). Inorder to synchronize the embryoid development, only the PEM fraction ofthe cell suspension with the size between 50 μm and 125 μm diameter wasused. This fraction was collected by using nylon sieves. When the PEMshad grown for seven days on OB5, the medium was refreshed to preventexhaustion of the nutrients and to eliminate single cells that did notdevelop into embryoids. Also in this stage of development differentamounts of ABA and sucrose were supplemented to the suspension. Therefreshing of the ABA-containing medium was repeated after another sevendays. The embryoids (torpedo stage) were harvested after a cultureperiod of 18 to 20 days on a 500 μm nylon sieve.

Desiccation and Germination

Before desiccation the embryoids were thoroughly rinsed with OB5 mediumin a Buchner-funnel with applied vacuum. Approximately 1 g of thefreshly harvested embryoids was transferred to a sterile plastic Petridish (9 cm) by forceps. The embryoids were equally spread out over thesurface of the Petri dish. The Petri dishes were covered and placed inhygrostats (Weges, 1987). Drying rates were varied by exposure todifferent relative humidities (RH) inside the hygrostat at 25° C.,generated by different saturated salt solutions with their RH betweenbrackets: Na₂ CO₃ (90%), NaCl (73% ), Ca(NO₃)₂ (50% ), CaCl₂ (30%) andLiCl (13%). Embryoids remained in the hygrostat until their moisturecontent was in equilibrium with the RH as measured by their weight loss.Rapid drying was effected by placing the Petri dishes without cover inthe air flow cabinet. The dry weight of the embryoids was determinedafter freeze drying for 24 h. The moisture content was calculated as gH₂ O/g dry weight (DW).

The viability (desiccation tolerance) of the embryoids was evaluatedwith a germination test. Approximately 100 dry embryoids were placed onfilter paper in a sterile plastic Petri dish (6 cm). Before imbibition,the embryoids inside the closed Petri dish were prehumidified inmoisture saturated air for four hours to prevent possible imbibitionaldamage (Hoekstra et al., 1989). Following this treatment, 1 ml OB5medium was provided to the embryoids. The Petri dish was sealed withParafilm and placed in an incubator with a 16 h/day photoperiod at 25°C. Embryoids were recorded as desiccation tolerant when they showedclear root growth within ten days.

Mode of Dehydration

In an attempt to regulate the drying rate in a repeatable manner, theembryoids were dried at different constant RHs (FIG. I). Rapidly driedembryoids were not able to germinate, whereas the embryoids, dehydratedslowly over a saturated Na₂ CO₃ solution turned brown and died beforethey reached their equilibrium moisture content. Maximum survival, 49%germination, was achieved when the embryoids were dried above asaturated CaCl₂ solution. The different drying treatments not onlyvaried in drying rate but also in final moisture content. In order tooptimize the results, the embryoids were exposed to a range of RHs thatwas decreased each fourth day. This method was based on Senaratna et al.(1989a) with slight modifications. Due to a suboptimal ABA concentration(3.8 μM) during the maturation phase, the germination could only beincreased with this method to maximally 76% (FIG. I).

In order to characterize the drying process, the moisture content ofembryoids grown under optimal maturation conditions, 37.9 μM ABA and 60g/l sucrose, was measured. Most of the water was lost in the first fourdays, but it took seven to nine days before they reached the equilibriummoisture content of 0.05 g H₂ O/g DW. To determine the desiccationtolerance in the course of slow drying, the embryoids were quicklydehydrated to a moisture content of 0.05 g H₂ O/g DW, at intervals.Germination increased to 100% on account of a slow drying treatment ofat least four days (FIG. 1).

Imbibition and Germination

To prevent imbibitional damage by too rapid a water uptake embryoidswere treated in a water vapour saturated atmosphere for differentlengths of time. FIG. 2 shows that germination improved with increasingprehydration time up to eight hours, after which germination decreased.As the embryoids lack endosperm, they might need additional nutritionfor proper regrowth. In Table II embryoids were germinated on B5 mediumor water. Without the nutrition, the embryoids germinated very poorly(4-5%), while with B5 medium the regrowth was optimal (98%). Potassiumleakage measurements revealed that embryoids imbibed in water with afour hour prehydration treatment leaked at a considerably higher ratethan those imbibed in B5 medium (data not shown).

Embryoid Maturation

Osmotic stress and ABA are the main parameters that play a role in theembryoid maturation. Therefore, concentrations of sucrose and ABA in thematuration medium were varied. ABA was supplemented to the medium oneweek after the start of the embryoid development. Earlier addition ofABA hindered embryoid development whereas too late an addition did notprevent precocious germination (data not shown). The concentration ofadded ABA had a clear effect on desiccation tolerance. Germinationreached its maximum between 19 and 37.9 μM ABA (FIG. 3). At higher ABAconcentrations desiccation tolerance was still high, but the yield oftorpedo-shaped embryoids decreased due to an impediment of developmentat earlier stages. At lower ABA concentrations desiccation tolerancedecreased. After imbibition only the roots elongated, while thehypocotyls and cotyledons turned brown.

The amount of reserves was estimated by measurements of the dry mattercontent. Embryoids grown without ABA had a much lower percentage dryweight than those grown with ABA addition, because they were alreadygerminating and had elongated roots and hypocotyls. Elevated ABAconcentrations hardly further increased the accumulation of dry matter(FIG. 4). The sucrose concentration had no effect on desiccationtolerance at the optimal ABA concentration of 37.9 μM, particularly notin genotype "Trophy" (Table III). But at 3.8 μM ABA high sucroseconcentrations (osmotic stress) had a positive effect on thegermination. Elevated sucrose levels increased the dry matter content ofthe embryoids (FIGS. 4 and 5). However, the higher sucroseconcentrations impeded embryoid development similarly as the elevatedABA concentrations, and the number of embryoids was reduced. The optimalsucrose concentration of the maturation medium for embryoid yield andregrowth performance was 60 g/l for "Trophy" and 20 g/l for "RS 1" (datanot shown).

Embryoid Development

Through subculturing at low density in 2,4-D-free B5 medium, PEMsdevelop into the subsequent embryogenic stages: globular, heart andtorpedo shape. The transition from undifferentiated to differentiatedgrowth is coincided with a decrease of moisture content (FIG. 6). The"RS 1" embryoids reached the torpedo stage after 10 days. The torpedoshaped embryoids have a moisture content of approximately 6 g H₂ O/g DW.Without ABA the embryoids then started to germinate precociously, whichcaused an increase in moisture content. When ABA (37.9 μM) was added thetorpedo embryoids continued their development, while their moisturecontent dropped to 3 to 4 g H₂ O/g DW. The decrease of the moisturecontent after 20 days of the ABA treated embryoids is due to the dryingtreatment over the saturated salt solutions. As a comparison themoisture content of carrot seeds during their development is also shownin FIG. 6.

Exposure of torpedo embryoids for three days to ABA was sufficient toinduce desiccation tolerance for genotypes "RS 1" and "Trophy" (FIGS. 7and 8). The germination percentage increased within this period for bothgenotypes to 100%. Globular- and heart-shaped embryoids, younger then 10days, never showed regrowth. Without ABA maximally 20% of the "RS 1"embryoids and 45% of the "Trophy" embryoids germinated after a slowdehydration to 0.05 g H₂ O/g DW moisture content. Exogenous ABA seemsnot to be the crucial factor, but it enhances the desiccation tolerancesignificantly. When the "Trophy" embryoids were cultured for more then11 days on ABA containing media the capacity to survive dehydrationdecreased, probably because they were producing secondary embryoids ontheir axes.

During production of desiccation tolerant carrot embryoids wedistinguish four subsequent phases; embryoid development, maturation,dehydration and germination. It is demonstrated in the presentdescription that 100% germination of rehydrated embryoids can only bereached when the importance of these four phases is recognized. Iida etal. (1992) reached 75% germination because they only optimized thematuration phase by varying the ABA treatment. Lecouteux et al. (1992)also claimed complete desiccation tolerance in carrot embryoids, withretention of viability for up to eight months at 4° C. However, theirembryoids still had a moisture content of 0.35 g H₂ O/g DW (25%) duringthe quiescent phase, which is far more than the usual moisture contentof seeds in storage (e.g. dry carrot seeds, 10% ). According to ourdefinition, these embryoids can not be called "desiccation tolerant".The embryoids might have been able to survive the storage period ofeight months, because of partial dehydration at low temperature, underconditions of which metabolism is much reduced.

Desiccation tolerance of embryoids has been reported also for otherplant species, but the methods described so far fail to attain 100%regrowth of the dried specimen (Anandarajah and McKersie, 1990 and 1991;Senaratna et al., 1989a,b; McKersie et al., 1989; Marsolais et al.,1991; Parrott et al., 1988; Roberts et al., 1990; Attree et al., 1991;Gray, 1990). The authors did not pay full attention to the foursubsequent phases in the embryoid production, which are discussed inmore detail hereafter.

Embryoid Development

For carrots it seems that only torpedo shaped embryoids, formed 7-10days after the start of the culture, are able to tolerate the dryingtreatment (FIGS. 7 and 8 ). The preceding embryogenic stages, globular-and heart shaped, were never desiccation tolerant. In a similar waybarley embryos acquired desiccation tolerance at a certain developmentalstage (16 days after pollination) and Arabidopsis embryos 12 days afterpollination (Bartels et al., 1988; Koornneef et al., 1989). Senaratna etal. (1989a and b) demonstrated with alfalfa that only torpedo andcotyledonary embryoids were able to germinate after desiccation. Iida etal. (1992) obtained with carrot similar results and suggested that onlythe torpedo embryoids were responsive to ABA. These data support theidea that somatic embryogenesis mimics zygotic embryogenesis.

Maturation

As soon as the histodifferentiation was completed the embryoids startedto mature. The maturation phase is characterized by the deposition oflipids, proteins and carbohydrates (reserves) (Kermode, 1990) and theacquisition of desiccation tolerance, while no apparent morphologicalchanges occur. Abscisic acid and osmotic stress play an important roleduring embryoid maturation. Both parameters are involved in theexpression of a specific set of genes and they both can inhibitprecocious germination (Kermode, 1990; Skriver and Mundy, 1990; Huet andJullien, 1992). From our experiments it is apparent that exogenous ABApromotes desiccation tolerance. However, without the addition of ABA asmall percentage of the embryoids still survived the drying treatment.This might indicate that induction of desiccation tolerance resides inthe developmental program of the embryoid and that it is not only due toexogenous ABA. Tolerance is lost when embryoids are switchedprecociously from the embryogenic program (maturation) to thegermination program, which will occur when ABA is left out from themedium. During a small time window just before embryoids may commenceprecocious germination, they are desiccation tolerant (FIGS. 7 and 8),probably because they contain some endogenous ABA (Iida et al., 1992).Also in a double mutant of Arabidobsis thaliana, lacking both ABAsynthesis and ABA sensitivity, some desiccation tolerance (15%) wasobserved during embryo development at 16 days after pollination(Koornneef et al., 1989). We only obtained 20% regrowth afterdehydration with genotype "RS 1". This percentage probably is so low,because of asynchronous embryoid development: some were still too young(heart shaped), others already proceeded into the germination phase. Inaccordance with the experiments by Huet and Jullien (1992), inhibitionof precocious germination by osmotic stress (60 g/l sucrose in thematuration medium) increased the percentage of desiccation tolerant"Trophy" embryoids to 45% in the absence of ABA (FIG. 8). In Table IIIwe also show that high sucrose concentrations (80-120 g/l, osmoticpressure -0.6 to -1.1 MPa) with low ABA concentration (3.8 μM) gavesimilar results as high ABA concentration (37.9 μM) without osmotictreatment, demonstrating that osmotic stress can replace ABA.Anandarajah and McKersie (1990 and 1991) were also able to inducedesiccation tolerance in somatic embryoids of alfalfa through elevatedsucrose concentrations. Also the vigour of the dry alfalfa embryoids wasenhanced, an effect that we have not noticed with our carrot embryoids.These data might be explained by an osmotically induced increase of theendogenous ABA concentration (Skriver and Mundy, 1990). However, it hasto be realized that sucrose not only acts as an osmoticum but also asthe sole carbohydrate source. Sucrose had a significant effect on thedry matter content of the embryoids (FIGS. 4 and 5). An optimalmaturation protocol apparently requires both ABA and a sucrosetreatment. This is further supported by the observation that embryoidsafter one week on ABA medium with 2% sucrose produced secondaryembryoids on their axes, thereby losing their desiccation tolerance.Embryoids grown in ABA at high sucrose concentrations never showedsecondary embryogenesis.

Dehydration

The rate of drying has been identified as a crucial factor during theacquisition of desiccation tolerance, not only in embryoids (McKersie etal., 1989; Seneratna et al., 1989a and b) but also in desiccationresistant nematodes (Madin and Crowe, 1975), slow drying appeared to beessential for survival. Carrot embryoids behaved similarly, only theslowly dried embryoids were able to germinate (FIG. 1). Theseobservations implicate that during the slow drying changes occur withinthe organisms which protect them from the deleterious effects ofdehydration. Resistant nematode species produce large amounts of thedisaccharide trehalose, that protects membranes and proteins in the drystate (Crowe et al.,1987). Also in plants large quantities of di- andoligo- saccharides are found in desiccation tolerant seeds (Koster andLeopold, 1988) and pollen (Hoekstra and Van Roekel, 1988). Not only thecarbohydrate content might change during dehydration but also theprotein content. Nordin et al. (1991) and Grossi et al. (1992) haverecently demonstrated that during drought stress a specific set of genesis expressed. Most of these genes are also induced by ABA, but some areexclusively expressed during drought stress. The resulting proteinsmight be crucial to survive desiccation stress. This suggestion mightalso explain why Iida et al. (1992) found such low germinationpercentages with their rapidly dried (3h) carrot embryoids. During thisshort drying period probably insufficient amounts of proteins andoligo-saccharides were synthesized for optimal regrowth.

Germination

The best way to measure desiccation tolerance is to determinegermination. Non-germinating embryoids are not necessarily desiccationintolerant, because germination can be hindered by dormancy or by thewrong germination procedure. Embryoids are naked, viz. not protected bya seed coat and endosperm, and therefore might be very sensitive toimbibitional damage and nutritional shortage. We have demonstrated thatprehydration significantly enhances regrowth (FIG. 2). The positiveeffect of prehydration on the germination percentage is an indicationthat stability of membranes may play a role in the desiccation toleranceof embryoids. Membrane phospholipids of dry organisms are in the gelphase (Hoekstra et al., 1989), which may also hold for dry embryoids.During imbibition the membrane changes from the gel phase to theliquid-crystalline phase. Such transition can cause leakage of cellsolutes when free water is available for solute transport, which may becatastrophic for the embryoid. Prehydration with moist air preventsleakage, because the transition then occurs in the absence of freewater. The lower germination percentage on water compared to B5 medium(Table II), might be explained by a lack of nutrition. However, theleakage measurements suggest that membrane integrity might play a rolehere as well. Tolerant embryoids imbibed in water leak at the same rateas intolerant embryoids in B5 medium.

Tolerance induction using LAB 173-711 (an ABA analog) The same set ofexperiments has been carried out with LAB 173-711 instead of ABA. Theresults are depicted in Table III.

                  TABLE III                                                       ______________________________________                                        Effect of LAB concentration on the desiccation tolerance of                     different organs of carrot embryoids. Embryoids were                          slowly dehydrated at controlled RH and prehydrated in                         moist air for four hours before imbibition in B5 medium.                                LAB (μM)                                                       regrowth    0     2       5    20     40  60                                  ______________________________________                                        roots       -     +       +    ++     ++  ++                                    shoots - - ± +  ++ ++                                                    ______________________________________                                         -= none;                                                                      ±= poor;                                                                   += fair;                                                                      ++= good                                                                 

These regrowth data are similar to those of embryoids treated with ABA.It can be concluded that LAB 173-711 is able to induce desiccationtolerance at the same concentration as ABA, although it is suggested tobe four times more effective than ABA.

Comparison of Zygotic and Somatic Embryogenesis

In FIG. 6 we have shown the development of seeds and embryoids of Daucuscarota on the basis of the moisture content. The curves look similar butthe main difference between the two types lies in the time that theembryo(id)s need to decrease the moisture content from 3 to 0.5 g H₂ O/gDW, which coincides with the maturation part of the development. Thezygotic embryo has a prolonged maturation phase with an extended reserveaccumulation as compared to the embryoid. We have to take into accountthat the data are from whole seeds, that is from embryos with endosperm.The condensed maturation of the embryoids might have reduced theregrowth potential, because the embryoids may not have been able tosynthesize all the necessary proteins, lipids and carbohydrates in the10-12 days maturation period. In contrast zygotic embryos have a 40-50days maturation phase. In this respect somatic embryogenesis does notmimic zygotic embryogenesis.

In conclusion our data clearly demonstrate that it is possible to inducecomplete desiccation tolerance in embryoids of different genotypes ofcarrot.

Tolerance Induction in Cucumber

We have got two lots of globular cucumber embryoids from Ahrensburg,which we have used to perform two ABA experiments. ABA (2-20 μM) addedto liquid or semi-solid B5 media with developing globular cucumberembryoids is also capable, like with carrot embryoids, to inhibit theprecocious germination. At too low ABA concentrations (0.01 1.0 μM)embryoids demonstrated radicle protrusion and greening of the"cotyledons". whereas the embryoids on high ABA media continued theirembryoids development to more or less torpedoshaped stages. The embryoiddevelopment on solid medium was clearly better than on liquid medium. Insuspension only very little cotyledons were formed, whereas embryoids onagar developed nice cotyledons. We also were able to dehydrate some ofthese cucumber embryoids and callus and the regrowth data are shown inTable II. Plant material was slowly dried and prehydrated in moist airbefore imbibition in B5 medium.

                  TABLE II                                                        ______________________________________                                        Effect ABA and sucrose on the development of desiccation                        tolerance in cucumher embryoids and callus.                                               ABA (μM)                                                     Sucrose (gr./1)                                                                             0     5          10   15                                        ______________________________________                                        20            -     -          -    -                                           60 - C/R C/R C/R                                                            ______________________________________                                         C: callus growth;                                                             R: root elongation                                                       

These data clearly demonstrate that also cucumber can acquiredesiccation tolerance and also ABA relatively high concentrations of arenecessary to survive dehydration to low moisture contents.

Coating Experiments

In order to avoid problems with rehydration of the embryoids and thuswith germination thereof coatings allowing for imbibation of theembryoids were developed.

Plant material: The same plant materials were used as described inmaterial and methods.

In this first experiment the basic coating consisted of parafinne 4444(Paramelt Syntac), with an additive coating consiting of sucrose.

    ______________________________________                                                 Coating mix:                                                                          A: 0% sucrose                                                   B: 0.5% sucrose                                                               C: 1% sucrose                                                                 D: 5% sucrose                                                                 E: 10% sucrose                                                                H: 50% sucrose                                                             ______________________________________                                    

Embryoids are imbibed in the melted coating mix (melting temperature 65°C.). They are harvested and cooled in ethanol or waters (understirring). The embryoids are (after a rest period) allowed to germinateat 25° C. in the light.

Results

    ______________________________________                                                plant           cool        germi-                                      number material coating sol. behav. nation remarks                          ______________________________________                                        01      Z4.8137 -       --          100%  control                               02 Z4.2973 -    75% control                                                   03 Z4.8137 A water float 0                                                    04 Z4.2973 A water float 0                                                    05 Z4.8137 B water float 0                                                    06 Z4.2973 B water float 0                                                    07 Z4.8137 C water float 0                                                    08 Z4.2973 C water float 0                                                    09 Z4.8137 D water float 0                                                    10 Z4.2973 D water float 0                                                    11 Z4.8137 E water float 0                                                    12 Z4.2973 E water float 0                                                    13 Z4.8137 H water sink 0                                                     14 Z4.2973 H water sink 0                                                   ______________________________________                                    

Cooling with alcohol did not work well because the drops formed"mushrooms" instead of "round pills". The cooling solution is probablyto apolar. Therefore only water was used as a cooling agent.

usage of a magnetic stirrer improves the shape of the pills, only whenthe droplets are sinking in the water. The shape might be improved if wecan make the cooling solution more polar through the addition of salts.

sucrose crystals did not distribute well through the coating mix,especially the large crystals. Therefore it was difficult to get ahomogenous coating.

Possible reasons for the lack of germination after the encapsulationmight be;

1: water supply is blocked through the wax, there are to less windowsfor sufficient water supply

2: lack of oxygen supply

3: heat shock of the treatment might damage the seeds

4: toxicity of the wax

Conclusion

The used coatings were impermeable for water, therefore no germinationoccurs. In order to improve the permeability for water of the coatingswe have to add more filling materials.

Breaking the Water Impermeability of the Wax Layer Through the Additionof Filling Materials

The same plant materials as in the previous experiment were used thesame basic coating was used. The additive coating was sucrose (grindedwith mortar mill) and/or Wimer 130® (Ankerpoort).

Coating Mix:

    ______________________________________                                        coat-    paraffine    wimer 130                                                                              sucrose                                          number (gr) (gr) (gr)                                                       ______________________________________                                        A002     2            1        0                                                B002 2 2 0                                                                    C002 2 3 0                                                                    D002 2 4 0                                                                    E002 2 1 1                                                                    F002 2 2 1                                                                    G002 2 3 1                                                                    H002 2 4 1                                                                  ______________________________________                                    

Cooling: water (no magnetic stirrer)

Melting temperature: 65° C. and 95° C.

Results

    ______________________________________                                        coat-number shape       behaviour remarks                                     ______________________________________                                        A002        half globule                                                                              float                                                   B002 half globule float                                                       C002 globular sink                                                            D002 nice globule sink                                                        E002 half globule float                                                       F002 nice globule sink                                                        G002 nice globule sink                                                        H002 rough globule sink very viscous                                        ______________________________________                                    

All structures were smooth on the outside when they were cooled inwater. The outside layer only contained wax and therefore the pills werestill water impermeable. When the droplets were cooled in ethanol theoutside also contained some filling materials but after imbibition inwater these crystals were released from the pill and a smooth waxy layerremained, with was again water impermeable. In order to lower theviscosity of the HOO2 coating we have increased the melting temp to 95°C., but this had not much effect, probably because of the high amount offilling materials. Possible solution; addition of a solvent (e.g.methanol)

Conclusion

The used coatings were still impermeable for water, because there wasstill to much wax at the outside of the pill. This was the reason toskip the seed encapsulation and germination test. In order to improvethe permeability for water of the coatings we have to lower the amountof wax by addition of other more polar materials.

Acquisition of water permeability of the coating layer through theaddition of PEG.

The same plant materials as in the previous experiments were used.

The basic coatings were paraffine 4444 and/or PEG 3400.

The additive coating comprised sucrose (grinded with mortar mill) and/orWimer 130®.

Coating mix:

    ______________________________________                                        coat-    paraffine PEG 3400  wimer 130                                                                             sucrose                                    number (gr) (gr) (gr) (gr)                                                  ______________________________________                                        A03 (D002)                                                                             2         0         4       0                                          B03 1.5 0.5 4 0                                                               C03 1 1 4 0                                                                   D03 0.5 1.5 4 0                                                               E03 0 2 4 0                                                                   F03 (H002) 2 0 4 1                                                            G03 1.5 0.5 4 1                                                               H03 1 1 4 1                                                                   I03 0.5 1.5 4 1                                                               J03 0 2 4 1                                                                   K03 0.5 1.5 0 0                                                               L03 0 2 0 0                                                                 ______________________________________                                    

Melting temperature: 65° C./81° C.

Results

    ______________________________________                                        coat-number    shape     behaviour remarks                                    ______________________________________                                        A03            droplets            liquid                                       B03 --   to viscous                                                           C03 --  to viscous                                                            D03 --  to viscous                                                            E03 --  viscous                                                               K03 droplets float liquid                                                     L03 droplets sink viscous                                                   ______________________________________                                    

Mixing PEG with wax was not successful when there is also Wimer 130 isadded, because the mix was not homogenous and top viscous to formdroplets. Without the filling material the mix of wax and PEG (K03) washomogenous and viscous, but still was able to form droplets. But duringthe cooling in water the materials separated, thereby the wax formed aimpermeable layer on the outside of the drops. Sometimes the PEG on theinside was wet and thus had gained water, which is undesirable. Whencooled in the air the Wax formed a layer on top of the PEG, because PEGis heavier than wax. Droplets of E03 and L03 contained of only polarmaterials and therefore dissolved easily in water. This might giveproblems with the rehydration of the embryoids. Water transport isprobably too easy in these coatings. To retard this water transportaddition of low amounts of wax or the addition of amphyphilic compoundslike fatty acids might work. Cooling in water is undesirable because thepill has to be somewhat permeable for water. When cooled in water theimbibition will start immediately. Water also promotes the apolarcompounds of the mix to settle on the outside thereby blocking the watertransport. The use of a apolar cooling liquid might be much better.

No encapsulation and germination test were performed because the mixesdid not fulfill our needs.

Conclusion

PEG can be used as a basic coating material, but we have to control thewater transport through the addition of wax or other apolar materials.Wax may not be desirable as basic coating material because it is toapolar and thereby restricts the water transport too much. The usedcoatings were still impermeable for water, because there was still toomuch wax at the outside of the pill. This was the reason to skip theseed encapsulation and germination test. In order to improve thepermeability for water of the coatings we have to lower the amount ofwax by addition of other more polar materials.

Restriction of water transport in coating based on PEG 3400 throughaddition of F 312 wax coating (Keyser and Mackay)

Basic coating: PEG 3400 (Harb/Heybroek).

Additive coating: F 312 wax coating (this was used instead of paraffine4444 because of its color.

Coating mix: 0.2%; 1%; 2%; 4%; 10%; 20% and 30% (K03) addition of F 312wax coating to the basic coating.

Cooling: air, water and sunflower oil

Melting temperature: 81° C.

Results

In all concentrations, except 30%, the wax formed droplets inside thePEG matrix. We hoped to get thinner wax layers on the outsine of thePEG. Only at 30% such a layer is formed but it is too thick. The watertransport was not restricted by these droplets, because all the pelletsdissolved very easily in water. Water could not be used as the coolingliquid. The oil worked well (only 20% mix was tested) but the congealingwas rather slow and therefore the whole pellet (in- and outside) wassoaked with oil. This might be profitable for the water transport, butit might give practical problems. The pellets cooled in oil dissolved inwater.

Conclusion

A mix of PEG with wax is not the solution for an optimal watertransport. It might be better to use materials that are less apolar thanwax, like fatty acids. Changing the polarity of the cooling liquid mightalso help in this case.

Formation of water permeable windows in apolar basic coating through theaddition of relatively light materials.

Basic coating: paraffine 4444 (0.9 gr/cm²) 4 gr

Additive coating:

Aerosil 200 (0.05 gr/cm²) 0.1 gr: A05

Dicalite 418 (0.21 gr/cm²) 0.4 gr: B05

Pumice 0-90μ (MCA) (0.90 gr/cm²) 2.7 gr: C05

Pumice 40-250μ (MCA) (0.90 gr/cm²) 2.7 gr: D05

cooling: water+tween,

alcohol+tween

melting temperature: 65° C.

Results

Pumice 0-90μ seems to be the best material for filling. We have testedthis by monitoring with the binocular the formation of air bubbles atthe surface of the pills. Because water goes into the pill while the airis pushed out. This does not mean that this is also the best for pillswith seeds. This is going to be tested in the next experiment. Pumice40-250μ behaved as a filling material with high density, that means thatit sinks to the bottom. No windows are formed in the outside layer ofthe pill. The other two materials had no effect during the air bubbletest, this might be due to the low amounts that were added to the basiccoating. Alcohol can also be used as cooling fluid, and has theadvantage that the outside of the pills are sterile. This might benecessary for the germination of embryoids.

Acquisition of water permeability of the coating layer through theaddition of lighter filling materials

Basic Coating: parffine 4444

Additive Coating: sucrose (grinded with mortar mill), Wimer 130(Ankerpoort), Pumice 0-90μ

Coating mix:

    ______________________________________                                        coat-    paraffine pumice    wimer 130                                                                             sucrose                                    number (gr) (gr) (gr) (gr)                                                  ______________________________________                                        A06*     2         2         1       0                                          B06* 2 1.5 2 0                                                                C06 2 0.5 3 0                                                                 D06* 2 0.5 4 0                                                                E06* 2 1.2 1 1                                                                F06 2 0.4 2 1                                                                 G06* 2 0.2 3 1                                                                H06* (H002) 2 0   4 1                                                       ______________________________________                                         *: indicates that the mix was diluted with acetone to improve the fluidit

Cooling: alcohol+Tween 20 (5.04.012) 2 droplets per 1000 ml

Melting temperature: 65° C.

Results

Dilution of the coating mix with acetone works very well during thepreparation of the pills. The mix forms better droplets because it isless viscous. The effect of the acetone on the seeds has still to beseen. The germination figures did not demonstrate any negative effect ofacetone. It even might be considered that addition of acetone has apositive effect on the germination of lettuce seeds, because it makesthe coating layer more open, which might promote water transport ormakes the coating easier to break through which the seeds can moreeasily grow out.

Germination test:

temp 25° C., light

    ______________________________________                                        coat-number  Z4.2973 Z4.8137     remarks                                      ______________________________________                                        control      90%     100%                                                       A06*  8%  92%.sup.1 perfect                                                      imbibition                                                                 B06* 16%  92%.sup.1 perfect                                                      imbibition                                                                 C06 33%  80%.sup.2 perfect                                                       imbibition                                                                 D06* 36% 100% perfect                                                            imbibition                                                                 E06*  0%  0% poor                                                                imbibition                                                                 F06  0%  0% poor                                                                 imbibition                                                                 G06*  0%  0% poor                                                                imbibition                                                                 H06* (H002)  0%  0% poor                                                         imbibition                                                               ______________________________________                                         .sup.1 : the pills that were scored as no germination contained seeds         which were germinated, but had not yet grown out of the coating.              .sup.2 : the pills that were scored as no germination contained seeds         which were indeed not germinated. These pills might be somewhat harder,       because no acetone was used.                                             

The poor imbibition of the seeds of the last four treatments was clearlyvisible, because the embryos were still glassy, whereas well imbibedembryos are white and tough. On the other hand the coating material ofthese pills seemed to contain more water than the first four treatments.This might be due to the sucrose which will attract water, but thisattraction inhibits the seed imbibition. We might have to look for othernon osmotic materials to serve as nutrition for the embryoids likestarch or proteins, or lower concentrations of sucrose. FIG. 9. showsthe course of water uptake of A06 and D06 pills without seeds. Thismeasurement was done in order to get an idea about the availability ofwater and water transport during the imbibition on filter paper. In thefuture this might be an easier method than a germination test to checkif the coating material satisfies our requirements.

Conclusion

We are able to create water permeability through the addition of lighterfilling materials, but we still have to test if the water transport isalso sufficient for the carrot embryoids. Measurement of the flux ofwater uptake might be very useful in this case.

The lower germination of the carrot seeds might be due to the toughcoating, whereas lettuce seeds are able to grow out of the pill.

Addition of sucrose to the coating completely inhibits the germinationof both types of seeds. The osmotic activity of sucrose causes the poorimbibition.

Reduction of water permeability of a coating mix based upon a watersoluble wax through the addition of apolar fatty acids. Monitoring theeffect of water soluble wax and cooling liquid on the germination ofcarrot and lettuce seeds.

Basic coating: PEG 3400

Additive coating: Pumice 0-90μ,

Wimer 130,

stearic acid (Merck),

palmitic acid (Merck)

cooling: sunflower oil+tween,

water+tween,

ethanol+tween,

methanol+tween

melting temperature: 65, 75, 85° C.

coating mix:

    ______________________________________                                                         wimer           palmitic                                                                             stearic                                 number PEG 3400 130 pumice acid acid                                        ______________________________________                                        E07    3 gr.     3 gr.    1 gr.  --     1 gr.                                   F07 3 gr. 3 gr. 1 gr. 1 gr. --                                              ______________________________________                                    

Results

E07 had to be melted at 75° C. because stearic acid only dissolved inthe mix at this higher temperature. To encapsulate the seeds the mix hadto be heated to 85° C. because at lower temperatures the mix congealedto fast. It was very difficult to make nice droplets, also at the highertemperature. This might give practical problems in the future. Insolublewax works much easier. F07 could only be encapsulated at 75° C., whereaspalmitic acid dissolved already at 65° C. in the mix.

Both mixes could be cooled with all four cooling liquids, but the fourcooling liquids all resulted in other kinds of pills. Especially thesurface and the shape were altered. The air bubble test showed thatwater was taken up by the pills independent of the cooling liquid. Itseemed that the pills made in ethanol gave more air bubbles, but thismust be confirmed with a water uptake test. We only tested F07 pillsmade in water (see FIG. 10) to get experience with the test and to seeif the test is accurate enough. The test worked out very well, but inthe case of a basic coating of PEG 3400 water uptake can not be measuredthrough weight increase. The pill looses weight because the basiccoating dissolves in the imbibition medium. In this case only thegermination test can tell if the pill satisfies our requirements.

Germination test:

temp 25° C., light

    ______________________________________                                        germination                                                                            plant                                                                coating  material cooling   4 days                                                                              7 days                                      ______________________________________                                        controle Z4.8137  --        100   100                                            Z4.2973 --  0  75                                                            E07 Z4.8137 alc. 100 100                                                        oil  0  72                                                                   Z4.2973 alc.  0  64                                                            oil  0  8                                                                   F07 Z4.8137 alc. 100 100                                                        oil  0  88                                                                   Z4.2973 alc.  0  75                                                            oil  0  45                                                                ______________________________________                                    

Additional results: the pills did not disintegrate during the imbibitionthrough the dissolvation of the basic coating in the water. When theseeds germinated the pills broke in two pieces.

Conclusions

From the germination data presented in the table we can draw thefollowing conclusions:

water soluble wax can be used as a basic coating. It does not inhibitthe germination, but there are some practical problems like theformation of nice droplets

the cooling liquid has a clear effect on the germination energy of theseed lots possibly caused through the regulation of water uptake.Cooling with oil gives the pill a more apolar character because some oilis absorbed by the coating mix. Therefore the coating might enhance theinhibition of the water uptake. But eventually the seeds becomecompletely imbibed and are able to germinate. It has to be tested whichcooling liquid gives the best regulation of water uptake for the carrotembryoids. Cooling with alcohol might result in a too rapid wateruptake.

the effect of the fatty acids in the mix is small, but it seems thatstearic acid gives better results.

Testing pumice 0-30μ (Profiltra) (a fine (0-25 μm) and light (0.9gr/cm3) filling material) for its water permeability in a wax coating.(comparison exp. 06)

Basic coating: paraffine 4444

Additive coating: Pumice 0-30μ,

Wimer 130,

acetone

coating mix:

    ______________________________________                                        coating                                                                            number  paraffine     pumice                                                                              wimer 130                                    ______________________________________                                        A08        7.5         5       0                                                B08 6 4 2                                                                     C08 5 3 4                                                                     D08 4 2 6                                                                     E08 4 1 8                                                                   ______________________________________                                    

To improve the fluidity of all the mixes aceton was added.

Cooling: ethanol+tween

Melting temperature: 65° C.

Results

Through the addition of more then 3 gr. Pumice 0-30μ to the mix, itbecame too solid. It was not possible to make the mix fluid withacetone, therefore we had to raise the amount of paraffine and still addacetone. Only A08 and E08 were used for water uptake profiles. Theseprofiles are made with 40 pills (+1.1 gr.). The water absorptionprofiles are shown in FIG. 22. There is not much difference between thetwo coating mixes. The pills still absorb water after 11 days.

Germination Test

temperature: 25° C., light

    ______________________________________                                        coating    Z4.8137            Z4 2973                                         number     4 days  7 days     4 days                                                                              7 days                                    ______________________________________                                        control    100%    100%       58%   84%                                         A08 50% 100% 0% 50%                                                           B08 25% 100% 0% 25%                                                           C08  8% 100% 0%  8%                                                           D08 16% 100% 0% 25%                                                           E08 (D06) 33% 100% 0% 16%                                                   ______________________________________                                    

All coating mixes give with both seed lots a retardation of thegermination. But in the case of Z4.8137 no effect was demonstrated onthe final germination percentage after 7 days, whereas for Z4.2973 itseems that the addition of Wimer 130 decreased the final germinationpercentage.

This is in contrast with the previous experiment, because there itseemed that addition of wimer 130 increased the germination. Thedifference between A08 and E08 (amount of Wimer 130) in the case ofZ4.2973, can not be explained by water transport, because E08 absorbedeven more water then A08 (see FIG. 11).

The final germination of coated Z4.2973 seeds never reached thegermination level of the control, despite the good rehydration of theseeds. It might be that oxygen supply is not sufficient, or the pillsmight be to tough.

Conclusion

Measuring water absorption profiles can be very useful for theunderstanding of the behaviour of coated seeds. Pumice 0-30μ can be usedfor filling material of waxy coatings

Testing other wax types in respect to paraffine 4444.

Plant material: Z4.8137, Z4.2973

Basic coating: Ozokerite® D306 (Keyser & Mackay) (57-59° C.),

Ciragref 80 slabs (Keyser & Mackay) (58-63° C.),

paraffine 2050/vk60 (Paramelt Syntac) (58-60° C.)

Additive coating: Wimer 130®,

Pumice

    ______________________________________                                        coating                           wimer                                         mix ozokerite ciragref paraffine 130 pumice                                 ______________________________________                                        A09      4                        6      0.5                                    B09  4  6 0.5                                                                 C09   4 6 0.5                                                               ______________________________________                                    

Cooling: water+tween,

methanol+tween,

ethanol+tween,

sunflower oil+tween

melting temp: 75° C.

Results

Ciragref did not fulfill our requirements, it was too viscous at 75° C.The other two waxes were perfect. They made very nice droplets in allcooling liquids. With the bubble test the pills cooled in ethanol gavethe best results.

Production of a dry embryoid pill with coating A08. Testing if carrotembryoid survive coating protocol.

Plant material: Dry carrot embryoids (cv "Trophy") produced in April1993 (experiment BT9, Wageningen). Dry embryoids were stored at 5° C.and 30% RH for more than 1,5 year.

Basic coating: paraffine 4444

Additive coating: Pumice 0-30μ (mix A08, see before) acetone

Melting temperature: 75° C.

Cooling liquid: ethanol+Tween

Storage: after the ethanol cooling the embryoid pills were collected ina sterile plastic petri-dish to dry. When they were dry (at least twohours) they were brought to a laminar air flow cabinet to be imbibed andgerminated under sterile (aseptic) conditions.

Germination: in a 6 mm petri dish with 3 autoclaved filter papers(Whatman nr 3) and 4.5 ml hormone free B5 medium with 20 gr./l sucrose(Gamborg et al, 1968). Temperature 25° C., in the light (cell 2 seedtech).

Germination was performed without a 4 hours prehydration treatment athigh relative humidity, which is necessary for imbibing naked embryoidsbecause of occurrence of imbibitional damage.

Results

Coating of embryoids was easier than seeds because they are muchsmaller. There were no problems with the sterility of the wholeprocedure, because no fungi or bacterial growth was found during thegermination test.

After 24 hours of imbibition, the embryoids were already coming out ofthe coating material (photographs 1-4) (FIG. 12). This is caused by theswelling of the embryoids,. It indicates that the coating materialpermeates B5 medium and it is not too tough to be pushed away by theswelling embryoids. The embryoids develop enough force to get out of thecoating. After 3 days of imbibition the first root elongation wasvisible (photographs 5-7) (FIG. 12). The embryoids were viable and hadsurvived the coating treatment. The next day the hypocotyls of theembryoids became green and the roots continued their elongation(photographs 8-11) (FIG. 12). After one week complete plantlets weredeveloped (photographs 12-13) (FIG. 12). The germination percentageafter one week was 50% (15/30).

Conclusion

It is possible to encapsulate dry carrot embryoids with a water freecoating layer without the loss of viability. With this coating layer thewater transport was in such a way that the prehydration treatment becameredundant.

BRIEF DESCRIPTION OF THE DRAWING

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon requesting payment of the necessaryfees.

FIG. 1: Effect of slow drying on germination of dry Daucus carotaembryoids (genotype "Trophy"). The embryoids were cultured for one weekon OB5 medium followed by four days on maturation medium with 37.9 μMABA and 60 g/l sucrose. At intervals during slow drying, the embryoidswere rapidly dried in sterile air for four hours to 0.05 g H₂ O/g DW.Before imbibition in OB5 medium, the embryoids were prehydrated in moistair for four hours. The moisture content data are the means±SD of fourreplicates, the germination data are the means±SD of duplicatemeasurements.

FIG. 2: Influence of duration of moist air pretreatment on thegermination of Daucus carota embryoids (genotype "Trophy"). Theembryoids were cultured one week on OB5 medium followed by four days onmaturation medium with 37.9 μM ABA and 60 g/l sucrose. The embryoidswere slowly dried for three days at 75%, 50% and 30% RH each, to amoisture content of 0.05 g H₂ O/g DW. The germination data are the means± SD of duplicate measurements.

FIG. 3: Effect of ABA concentration on the desiccation tolerance ofDaucus carota embryoids (genotype "RS 1") after 20 days in culture. ABAwas added on the seventh day of the culture. The embryoids weresuccessively dried for three days at 75%, 50% and 30% RH, to a moisturecontent of 0.05 g H₂ O/g DW. Before imbibition in OB5 medium, theembryoids were prehydrated in moist air for four hours. The germinationdata are the means±SD of two or four replicates.

FIG. 4: Effect of ABA on dry matter content of Daucus carota embryoids(genotype "RS 1") after 20 days in culture. The embryoids were culturedon OB5 medium. On day 7 the embryoids were transferred to mediacontaining different ABA concentration and either 20 g/l (-O-) or 60 g/lsucrose (- -). The data are the means±SD of triplicate measurements.

FIG. 5: Effect of sucrose concentration on dry matter content of Daucuscarota embryoids after 20 days in culture. The embryoids were culturedon OB5 medium. On day 7 the embryoids were transferred to mediacontaining different sucrose concentration and either 3.8 (-O-) or 37.9μM ABA (- -). The data are the means±SD of triplicate measurements.

FIG. 6: Changes in moisture content during development of Daucus carotaembryoids (genotype "RS 1"), with or without ABA, and of seeds of thesame species. ABA (37.9 μM) was added on the seventh day of the culture.After 20 days the embryoids, treated with ABA, are slowly dried abovesaturated salt solutions as described in FIG. 2. The seed moisturecontent data are redrawn from Gray and Steckel (1982).

FIG. 7: Influence of ABA on the development of desiccation tolerance ofDaucus carota embryoids (genotype "RS 1"). Embryoids were grown on B5medium with 20 g/l sucrose throughout the culture period. On day 7 theembryoids were transferred to fresh B5 media either without (-O-) orwith 37.9 μM ABA (- -). The embryoids were removed from the media afterthe indicated cultivation periods. Before germination embryoids wereslowly dried. See FIG. 3 for description of slow drying and germinationmethod.

FIG. 8: Influence of ABA on the development of desiccation tolerance ofDaucus carota embryoids (genotype "Trophy"). Embryoids were cultured thefirst week on B5 medium with 20 g/l sucrose and afterwards on B5 mediumwith 60 g/l sucrose. On day 7 the-embryoids were transferred to fresh B5media either without (-O-) or with 37.9 μM ABA (- -). The embryoids wereremoved from the media after the indicated cultivation periods. Beforegermination embryoids were slowly dried. See FIG. 3 for description of aslow drying and germination method.

FIGS. 9A and 9B show the course of water uptake of A06 and D06 pillswithout seeds.

FIGS. 10A and 10B show the relationship between water uptake andimbibition time for coating F07 as described in the experiments.

FIG. 11 shows the relationship between water uptake and imbibition timefor coatings A08 and E08 as described in the experiments.

FIG. 12 shows photographs 1-10 giving the swelling of dried toleranceinduced carrot embryoids coated as described in the last of the coatingexperiments.

We claim:
 1. Method for the induction of essentially complete desiccation tolerance in plant embryoids including the steps of treating the embryoids with an amount of abscisic acid activity which is at least about 110% about the amount used to induce quiescence, and coating the embryoids with a mixture of an apolar material and a polar hygroscopic material.
 2. Method according to claim 1, comprising a drying step after the treatment with abscisic acid activity.
 3. Method according to claim 2, wherein the drying rate of the embryoids is between 0.01 g H₂ O/g dry weight per hour and 1 g H₂ O/g dry weight per hour.
 4. Method according to claim 1, wherein the embryoids used are in the torpedo stage.
 5. Method according to claim 1 whereby the abscisic acid activity is produced at least partly in situ.
 6. Method according to claim 5, wherein the abscisic acid activity is induced by stress.
 7. Method according to claim 6, whereby the stress is provided through a heat shock.
 8. Method according to claim 6, whereby the stress is provided through a low temperature treatment.
 9. Method according to claim 6, whereby the stress is osmotic stress.
 10. Method according to claim 9, wherein the osmotic stress is provided by a carbohydrate or a polymer.
 11. Method according to claim 9, wherein the osmotic stress is provided at a level of -0.5 to -2.5 mPa.
 12. Method according to claim 1 wherein exogenous abscisic acid activity is added.
 13. Method according to claim 12, wherein the total absisic acid activity is 110-1000% of the activity used to induce quiescence.
 14. Method according to claim 12, wherein the exogenous abscisic activity is provided as abscisic acid.
 15. Method according to claim 12, wherein the exogenous abscisic acid activity is provided by at least one abscisic acid analog.
 16. Method according to claim 1, wherein the treated embryoids are carrot embryoids.
 17. Method for germination of embryoids according to claim 16, whereby the embryoids are prehydrated.
 18. Method according to claim 17 wherein the prehydration is carried out for 2-8 hours at 100% relative humidity and room temperature.
 19. Method according to claim 1, wherein the inorganic material is pumice and the wax-like material is parrafine.
 20. Method for the germination of embryoids according to claim 16, whereby the embryoids are imbibed at a temperature of 25-50° C.
 21. Method for the germination of embryoids according to claim 16 whereby the embryoids are imbibed in a medium comprising di- or trisaccharides.
 22. Method according to claim 16 wherein the abscisic acid activity is present in an amount equivalent to the activity of abscisic acid in the range of 1-100 μM. 